Benzo[b]thiophenesulphonamide 1,1-dioxide derivatives inhibit tNOX activity in a redox state-dependent manner.

Benzo[b]thiophenesulphonamide 1,1-dioxide (BTS) derivatives are strong cytotoxic agents that induce reactive oxygen species (ROS) overproduction and apoptosis in tumour cells. Although the precise origin of BTS-induced ROS is not known, a clear correlation between their cytotoxic effect and ability to inhibit a tumour-associated NADH oxidase (tNOX) activity of the plasma membrane has been described. To analyse the putative implication of tNOX in BTS-induced ROS generation, in this work we have synthesised and tested a new BTS derivative, the 6-[N-(2-phenylethyl)]benzo[b]thiophenesulphonamide 1,1-dioxide. According to its high lipophilicity, this compound showed a strong cytotoxic activity against a panel of six human tumour cell lines, including two human leukaemia (K-562 and CCRF-CEM) and four human solid tumours (HT-29, HTB54, HeLa and MEL-AC). We also tested the ability of this compound to inhibit the tNOX activity and we found an absolute dependence of this inhibition on the redox state of the tNOX: while under reducing conditions, that is, 100 mM GSH, the drug inhibits strongly the NOX activity with an EC(50) of about 0.1 nM, under oxidising conditions, there is no effect of the drug or just a slight stimulation of activity.

Synthesis and cytotoxic activity of lipophilic sulphonamide derivatives of the benzo[b]thiophene 1,1-dioxide.

In the search of new compounds with antineoplastic activity, we have analysed the effect of several structural modifications on the nucleus 6-benzo[b]thiophenesulphonamide 1,1-dioxide on its cytotoxic activity on tumour cells. Lipophilic substituents on the sulphonamide group significantly increased the cytotoxic activity measured using a panel of human tumour cell lines. Only slight variations on cytotoxicity were obtained when the sulphonamide group occupied the position 5 of the system. The most active compound was the N-4-methoxyphenyl derivative 15, which showed GI(50) values of 1-9 nM against HT-29, CCRF-CEM, K-562 and MEL-AC cells and of 200 nM against HTB-54 cells. Free access to the 3-position of the heterocyclic system seems to be required to obtain cytotoxic derivatives. Derivative 15 was also active at the same level of commercial Doxorubicine against cultured normal human lung fibroblasts.

New benzo(b)thiophenesulphonamide 1,1-dioxide derivatives induce a reactive oxygen species-mediated process of apoptosis in tumour cells.

In this work, we describe the process of cell death induced by a series of new benzo(b)thiophenesulphonamide 1,1-dioxide derivatives (BTS) that have been selected as candidate antineoplastic drugs. Human leukaemic CCRF-CEM cells incubated with BTS undergo a typical apoptotic process that includes cell shrinkage, phosphatidylserine translocation to the cell surface, mitochondrial dysfunction, caspase activation, chromatin condensation and internucleosomal DNA degradation. Mitochondrial alterations included dissipation of the mitochondrial membrane potential, oxidation of the phospholipid cardiolipin, release of cytochrome c and uncoupling of the mitochondrial respiratory chain, leading to a decrease of the intracellular ATP pool. Activation of caspase-8, -9 and -3 takes place during BTS-induced apoptosis. Either the addition of the specific caspase-8 inhibitor Z-IETD-fmk, or the overexpression of the antiapoptotic protein Bcl-2 significantly prevented BTS-induced apoptosis, suggesting the involvement of both caspase-8-regulated and mitochondria-dependent signalling pathways in this process. BTS induce a significant increase in the production and accumulation of intracellular reactive oxygen species (ROS) that can be observed within minutes after drug addition. Moreover, cytochrome c release, caspase-3 activation and cell death can be completely abrogated by a previous incubation with the antioxidant N-acetyl-cysteine. These results suggest that ROS are essential mediators in BTS-induced apoptosis.

New cytotoxic benzo(b)thiophenilsulfonamide 1,1-dioxide derivatives inhibit a NADH oxidase located in plasma membranes of tumour cells.

A series of benzo(b)thiophenesulfonamide 1,1-dioxide derivatives (BTS) have been designed and synthesized as candidate antineoplastic drugs. Several of these compounds have shown in vitro cytotoxic activity on leukaemic CCRF-CEM cells. The cytotoxic BTS, but not the inactive ones, were able to inhibit a tumour cell-specific NADH oxidase activity present in the membrane of CCRF-CEM cells.

Synthesis and cytotoxic activity of N-(2-pyridylsulfenyl)urea derivatives. A new class of potential antineoplastic agents.

Starting from a 3D-model for the antineoplastic activity of diarylsulfonylureas several new features were proposed and tested. Both types of assayed compounds, the N-(2-pyridylsulfonyl)urea and N-(2-pyridylsulfenyl)urea derivatives, inhibited by 50% the growth of the CCRF-CEM cell line at a dosage near to 1 microM. The N -(2-pyrimidinyl) derivative of the sulfenylurea 6c showed a better profile against HT-29, K-562 and HTB-54 tumor cell lines than the corresponding sulfonylurea 6b. Structural modifications on aryl systems affected differently to the cytotoxic activity shown by the compounds against each cell line.

Molecular cloning and characterization of the human p44 mitogen-activated protein kinase gene.

The complete genomic structure of the human p44(mapk) gene (HMGW-approved symbol PRKM3) has been determined. The gene covers 9 kb and is composed of nine exons and eight introns. This structure is identical to the previously reported mouse p44(mapk) gene, indicating a high degree of evolutionary conservation. A sequence differing by one nucleotide from the consensus TATA box is present 132 positions upstream of the main transcription initiation point. This point has been located 415 nucleotides upstream of the translation initiation codon ATG and perfectly meets the consensus criteria for an initiator element (Inr). Multiple consensus sequences for factors that regulate either basal transcription or gene expression during cell differentiation and proliferation can be found in the putative promoter region. Some of them, such as several G/C boxes located downstream from the transcription initiation point, are also present in the homologous mouse gene, where they were shown to be functional.

cAMP activates transcription of the human glucocorticoid receptor gene promoter.

Glucocorticoids and cAMP regulate, either in a synergistic or additive fashion, the transcription of multiple genes, although some antagonistic effects of dexamethasone on cAMP-activated transcription have been described. The increased glucocorticoid receptor (GR) mediated response of some cell types, as a result of augmented cAMP, has been considered to be mainly due to an increased stability of GR mRNA, although other plausible explanations should not be ruled out. We studied the possibility that GR transcription itself could be affected by cAMP levels. HeLa cells were transfected with human GR (hGR) promoter constructs and their transcriptional activity determined after inducing a cAMP increase with forskolin. We found that forskolin almost doubled the transcriptional activity of the promoter construct spanning -2995 to +38 of the hGR, whereas no significant variations were observed with shorter chimeras containing sequences downstream -979. Shift mobility showed binding of CREB in vitro to a putative cAMP responsive element located at -1000, suggesting that hGR may be upregulated by cAMP at the transcriptional level, thus adding a new mechanism ascribable to this second messenger, which in conjunction with the cAMP-induced GR mRNA increased stability, would lead to a more precise control of the amount of GR protein within the cell.

Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene.

The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. We studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene alpha, along with their 5'- and 3'-flanking regions, we identified a 4-base deletion at the 3'-boundary of exon 6 in one GR allele (delta 4), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G1220) in the proband, in one of her affected brothers and in her unaffected sister. The functional importance of this mutation was tested in a cotransfection study using the recombinant expression vector pRShGR-Ser363 and the glucocorticoid responsive vector mouse mammary tumor virus-chloramphenicol transferase. This amino acid substitution did not alter the function of the glucocorticoid receptor. Using reverse transcription-polymerase chain reaction we could only identify messenger RNA transcripts of the G1220-allele but not of the delta 4-allele in the affected members of this family who were heterozygous for the G1220 mutation. This deletion in the glucocorticoid receptor gene was, thus, associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism.

A systematic search for a bipolar predisposing locus on chromosome 5.

Chromosome 5 markers spanning the pter to the qter were used to examine linkage to bipolar illness in 14 pedigrees. Twenty-four loci were examined in 237 individuals, of whom 69 were either bipolars or schizoaffectives. Marker genotypes were determined for each individual and lod scores were calculated under a dominant disease model with a maximum penetrance of 85%, a disease gene frequency of 0.015, a variable age of onset, and a phenocopy rate of 0.001. Under the assumption that bipolar illness is genetically homogeneous, the total lod scores from all pedigrees with each marker were uniformly lower than -2.0, suggesting the absence of linkage to disease at any of these loci. Multipoint analysis allowed exclusion of intervals between markers. When lod scores were calculated allowing for heterogeneity, no subset of linked families was found. These results indicate that in our pedigree series almost the entire mapped region of chromosome 5 can be excluded for linkage to bipolar illness.

The genomic structure of the human glucocorticoid receptor.

We have determined the structure of the human glucocorticoid receptor (hGR) gene after the isolation and characterization of cosmid clones mapping to discrete regions of the cDNA. The gene contains a total of 10 exons and has a minimum size of 80 kilobases. Exon 1 consists solely of 5'-untranslated sequence, and exon 2 encodes the amino-terminal portion of the receptor. The two putative zinc fingers are separately encoded by two exons, and a total of five exons combine to form the cortisol-binding domain. By restriction mapping and sequence analysis of cosmids located on the 3'-end of the gene, we have established that the two receptor isoforms, hGR alpha and hGR beta, originate from the same gene by alternative splicing. Each hGR isoform is encoded by nine exons, of which the first eight are identical, whereas the ninth exons are heterologous. Multiple GC boxes and no obvious TATA or CAAT elements have been found in the 5'-flanking region. S1 nuclease analysis yielded one major band, and the transcription start site is localized to the *C residue within TAC*CCTC. Alignment of sequences around the splice junctions of hGR with those of other members of the steroid receptor superfamily revealed three different splice positions within the DNA-binding domain. This comparison also permitted the prediction of the positions of the splice sites and the sizes of the putative exons in the human mineralocorticoid receptor.

Effect of reducing agents and uncouplers on the electrical potential generated by mitochondrial ATPase activity.

Beef heart submitochondrial particles bound to phospholipids impregnated filters generated an electrical potential upon the addition of ATP. The magnitude of the electrical potential reached depended on the phospholipid mixture composition used for filter impregnation, phosphatidylethanolamine being the active component for the electrical potential generation. Uncoupler FCCP (p-trifluoromethoxy carbonyl cyanide phenylhydrazone) inhibited the transmembrane electrical potential generation by diminishing the electrical resistance of the system as a result of its protonophoric action. However, uncouplers 2, 4-dinitrophenol and dicoumarol did not provoke large modifications of the electrical resistance under the conditions of pH and concentration used, and their action varied with the time elapsed after the submitochondrial particles purification, favouring the idea of the uncoupler interaction with a specific site on the membrane. Addition of sodium dithionite resulted in a higher plateau value for the electrical potential consistent with the promoted increase in ATPase activity. The effect of this agent was reversed by the 2,6-dichlorophenol-indophenol added at equivalent concentrations.

Exclusion of linkage to 5q11-13 in families with schizophrenia and other psychiatric disorders.

Recently a linkage study on five Icelandic and two English pedigrees has provided evidence for a dominant gene for schizophrenia on 5q11-13 (ref. 1). In that study, families with bipolar illness were not included. Using the same probes, two similar but independent investigations on one Swedish pedigree and on fifteen Scottish families excluded linkage to schizophrenia. To evaluate whether the susceptibility gene on 5q11-13 is a common cause of schizophrenia in other populations, we examined five affected North American pedigrees using probes to the D5S39, D5S76 and dihydrofolate reductase loci. Two families in the present series had cases of bipolar disorder. We found that linkage can be excluded by multipoint analysis. These results, taken together, suggest that the disease gene on 5q11-13 does not account for most cases of familial schizophrenia.

Interaction of F1-ATPase and its inhibitor peptide. Effect of pH.

1. The inhibition of F1-ATPase by its natural peptide inhibitor is mixed non-competitive with two pH optimum values (5.5 and 8.2). 2. A two-step model for the interaction is suggested in which two enzyme conformations would exhibit different affinities for the peptide. 3. At low pH, interaction would be favoured. At high pH, a conformation (not susceptible to inhibition) changes into another (susceptible to inhibition) through the hydrolytic reaction stimulation, due to high pH.

Interaction of F1-ATPase and its inhibitor peptide. Effect of dinitrophenol, nucleotides and anions.

1. ATPase natural inhibitor interacted in a mixed non-competitive manner with compounds affecting hydrolytic activity. 2. Ka's for DNP, HCO3- and free ATP, and Ki's for SCN- and ADP became smaller as inhibitor peptide concentration increased, reflecting an increase in affinity of F1-ATPase for these compounds induced by the peptide. 3. Activators increased the peptide inhibitory effect, whereas inhibitors decreased it. 4. A two-step model for the peptide-enzyme interaction is suggested in which ATP hydrolysis is a key factor.